A New Quinazolinone Alkaloid along with Known Compounds with Seed-Germination-Promoting Activity from Rhodiola tibetica Endophytic Fungus Penicillium sp. HJT-A-6

A new quinazolinone alkaloid named peniquinazolinone A (1), as well as eleven known compounds, 2-(2-hydroxy-3-phenylpropionamido)-N-methylbenzamide (2), viridicatin (3), viridicatol (4), (±)-cyclopeptin (5a/5b), dehydrocyclopeptin (6), cyclopenin (7), cyclopenol (8), methyl-indole-3-carboxylate (9), 2,5-dihydroxyphenyl acetate (10), methyl m-hydroxyphenylacetate (11), and conidiogenone B (12), were isolated from the endophytic Penicillium sp. HJT-A-6. The chemical structures of all the compounds were elucidated by comprehensive spectroscopic analysis, including 1D and 2D NMR and HRESIMS. The absolute configuration at C-13 of peniquinazolinone A (1) was established by applying the modified Mosher’s method. Compounds 2, 3, and 7 exhibited an optimal promoting effect on the seed germination of Rhodiola tibetica at a concentration of 0.01 mg/mL, while the optimal concentration for compounds 4 and 9 to promote Rhodiola tibetica seed germination was 0.001 mg/mL. Compound 12 showed optimal seed-germination-promoting activity at a concentration of 0.1 mg/mL. Compared with the positive drug 6-benzyladenine (6-BA), compounds 2, 3, 4, 7, 9, and 12 could extend the seed germination period of Rhodiola tibetica up to the 11th day.


Introduction
Significant mutualistic relationships have been established between endophytic fungi and their host plants, attracting considerable attention due to their ecological and biotechnological potential [1].Endophytic fungi can produce a variety of secondary metabolites on their own and can be involved in the biosynthesis and biotransformation of secondary metabolites in host plants, making them an important source of active natural products.Active natural products derived from endophytic fungi hold vast potential applications in biopharmaceuticals, agricultural production, and industrial fermentation [2][3][4][5].Currently, the secondary metabolites isolated from endophytic fungi fermentation products include alkaloids, polyketides, terpenes, etc. [2,6], and some even possess activity in promoting seed germination [7].
Herein, as part of our ongoing studies on the bioactive secondary metabolites from Rhodiola tibetica endophytic fungi [20][21][22], we performed a Global Natural Products Social (GNPS) molecular networking analysis of the EtOAc extract of the endophytic fungus Penicillium sp.HJT-A-6.GNPS molecular networking has been widely applied in the analysis of natural products to cluster compounds with similar MS/MS spectra, expediting the dereplication process of known natural products [23][24][25].The GNPS molecular networking analysis of the EtOAc extract led to the discovery of a new quinazolinone alkaloid, peniquinazolinone A (1), together with eleven known secondary metabolites (Figure 1).The seed-germination-promoting activities of the isolated compounds were also evaluated.
Herein, as part of our ongoing studies on the bioactive secondary metabolites from Rhodiola tibetica endophytic fungi [20][21][22], we performed a Global Natural Products Social (GNPS) molecular networking analysis of the EtOAc extract of the endophytic fungus Penicillium sp.HJT-A-6.GNPS molecular networking has been widely applied in the analysis of natural products to cluster compounds with similar MS/MS spectra, expediting the dereplication process of known natural products [23][24][25].The GNPS molecular networking analysis of the EtOAc extract led to the discovery of a new quinazolinone alkaloid, peniquinazolinone A (1), together with eleven known secondary metabolites (Figure 1).The seed-germination-promoting activities of the isolated compounds were also evaluated.

Molecular Networking-Guided Isolation Workflow
To target the isolation of the quinazolinone alkaloid, the crude EtOAc extract of Penicillium sp.HJT-A-6 was subjected to a full-scan HPLC-Q-TOF-MS/MS analysis.The obtained MS/MS data were used to generate the initial molecular network using the GNPS platform following the established protocol.This initial network was visualized and analyzed via Cytoscape 3.7.1 software.
As shown in Figure 2, the annotated nodes of the total secondary metabolites are displayed; the color of the node indicates the m/z of the parent ion.This process generated a network featuring 2373 nodes and 755 clusters; those representative highlighted clusters

Molecular Networking-Guided Isolation Workflow
To target the isolation of the quinazolinone alkaloid, the crude EtOAc extract of Penicillium sp.HJT-A-6 was subjected to a full-scan HPLC-Q-TOF-MS/MS analysis.The obtained MS/MS data were used to generate the initial molecular network using the GNPS platform following the established protocol.This initial network was visualized and analyzed via Cytoscape 3.7.1 software.
As shown in Figure 2, the annotated nodes of the total secondary metabolites are displayed; the color of the node indicates the m/z of the parent ion.This process generated a network featuring 2373 nodes and 755 clusters; those representative highlighted clusters indicated several known natural products and a promising new compound.In cluster 1, highlighted in light red, the node m/z 276.062 was connected to the node m/z 260.069, which were supposed to be two known compounds, viridicatin (3) and viridicatol (4).Clusters 2 and 3, highlighted in light blue and light purple, respectively, were used to identify two classes of natural products, cyclopeptin analogues (5a/5b-7) and phenyl acetate derivates (10,11).The orphan cluster 4 (m/z 247.144) in light green, which was not associated with any structurally known compound, indicates the presence of an unidentified compound.Thus, using m/z 247.144 as a guide, a new quinazolinone alkaloid, peniquinazolinone A (1), was isolated, along with known compounds 2-12.
indicated several known natural products and a promising new compound.In cluster 1, highlighted in light red, the node m/z 276.062 was connected to the node m/z 260.069, which were supposed to be two known compounds, viridicatin (3) and viridicatol (4).Clusters 2 and 3, highlighted in light blue and light purple, respectively, were used to identify two classes of natural products, cyclopeptin analogues (5a/5b-7) and phenyl acetate derivates (10,11).The orphan cluster 4 (m/z 247.144) in light green, which was not associated with any structurally known compound, indicates the presence of an unidentified compound.Thus, using m/z 247.144 as a guide, a new quinazolinone alkaloid, peniquinazolinone A (1), was isolated, along with known compounds 2-12.

Seed-Germination-Promoting Activity of the Isolated Compounds
Compounds 2, 3, 4, 7, 9, and 12 were tested for their seed-germination-promoting activity.As shown in Figure 5, compounds 2, 3, and 7 showed an optimal promoting effect on the seed germination of Rhodiola tibetica at a concentration of 0.01 mg/mL, with a germination rate of about 62%, 70%, and 62%, respectively, compared to the germination rate of about 52% of the positive drug 6-BA.Compounds 4 and 9 exhibited a germination rate of about 72% and 58%, respectively, at the optimal concentration of 0.001 mg/mL, indicating a negative correlation between concentration and germination rate.Compared with other tested compounds, compound 12 showed the best germination rate of about 62% at the same concentration level (0.1 mg/mL).In addition, compounds 2, 3, 4, 7, 9, and 12 could delay the seed germination of Rhodiola tibetica up to the 11th day, while the positive drug 6-BA only affected the seed germination process until the 9th day (Figure 6).Compounds 2, 3, 7, and 12 did not show concentration-dependent activity, which probably activated the upregulation of certain genes responsible for seed germination at concentrations ranging from 0.001 to 0.01 mg/mL.When the concentration exceeded a certain level, the expression levels of the genes responsible for seed germination decreased, hence not exhibiting concentration dependence.Additionally, the expression levels of certain genes responsible for seed germination were also influenced by the duration of time.Compound 12, however, exhibited the opposite behavior.
Compound 1 was not tested for its seed-germination-promoting activity due to the trace amount.Compound 2 is a structural analogue of compound 1, generated by the ring opening of the quinazoline moiety of compound 1; further seed germination assays of compound 1 may verify whether the existence of the quinazoline moiety affects its seedgermination-promoting activity.

Seed-Germination-Promoting Activity of the Isolated Compounds
Compounds 2, 3, 4, 7, 9, and 12 were tested for their seed-germination-promoting activity.As shown in Figure 5, compounds 2, 3, and 7 showed an optimal promoting effect on the seed germination of Rhodiola tibetica at a concentration of 0.01 mg/mL, with a germination rate of about 62%, 70%, and 62%, respectively, compared to the germination rate of about 52% of the positive drug 6-BA.Compounds 4 and 9 exhibited a germination rate of about 72% and 58%, respectively, at the optimal concentration of 0.001 mg/mL, indicating a negative correlation between concentration and germination rate.Compared with other tested compounds, compound 12 showed the best germination rate of about 62% at the same concentration level (0.1 mg/mL).In addition, compounds 2, 3, 4, 7, 9, and 12 could delay the seed germination of Rhodiola tibetica up to the 11th day, while the positive drug 6-BA only affected the seed germination process until the 9th day (Figure 6).Compounds 2, 3, 7, and 12 did not show concentration-dependent activity, which probably activated the upregulation of certain genes responsible for seed germination at concentrations ranging from 0.001 to 0.01 mg/mL.When the concentration exceeded a certain level, the expression levels of the genes responsible for seed germination decreased, hence not exhibiting concentration dependence.Additionally, the expression levels of certain genes responsible for seed germination were also influenced by the duration of time.Compound 12, however, exhibited the opposite behavior.Compound 1 was not tested for its seed-germination-promoting activity due to the trace amount.Compound 2 is a structural analogue of compound 1, generated by the ring opening of the quinazoline moiety of compound 1; further seed germination assays of compound 1 may verify whether the existence of the quinazoline moiety affects its seed-germination-promoting activity.

Fungal Material
The fungus strain Penicillium sp.HJT-A-6 was obtained from the stem of Rhodiola tibetica collected in Langkazi County, Shannan City, Tibet, China, in July 2021.It was identified based on its morphological characteristics and its sequence in the internal transcribed spacer (ITS) analysis of rDNA, and the BLAST search result showed that the sequence was the most similar (99%) to the sequence of Penicillium sp.(compared to MN634462.1).The sequence data of the fungus were submitted to the GenBank database, accession number: OR346333.1.The fungus was deposited in the College of Life and Health, Dalian University, Dalian, China.

Fermentation and Isolation
The fungal strain was cultured on an autoclaved rice medium (one hundred 500 mL Erlenmeyer flasks, each containing 80 g rice, 110 mL water) in the stationary phase at 28 °C for 40 days.After 40 days, the fermentation material was cut into small pieces and extracted with 95% EtOH three times.The extract was concentrated under reduced pressure to afford an aqueous solution and then partitioned with petroleum ether, EtOAc and n-BuOH to obtain the EtOAc-soluble extract (64 g).The extract was subjected to silica gel column chromatography with CH 2 Cl 2 /MeOH (100:0-0:100) to afford fourteen fractions (Fr.A-N).

Seed-Germination-Promoting Assay
Compounds 2, 3, 4, 7, 9, and 12 were dissolved in a 0.2% DMSO aqueous solution to yield a stock solution with a concentration of 0.06 mg/mL.Then, 5 µL, 50 µL, and 500 µL of compounds 2, 3, 4, 7, 9, and 12 were added to a 30 mm filter paper placed in a 6-well plate.After the evaporation of the solvent, the filter paper was immersed in 300 µL of distilled water, and then 20 seeds of Rhodiola tibetica were displayed in each 30 mm filter paper and incubated with a light-dark regime of 16:8 h at 20 • C for 7 days.The germination rate of the seeds was calculated after incubation.The experimental data were collected from three independent experiments.Further experiments on the relationship between the number of germinated seeds and germination time for compounds 2, 3, 4, 7, 9, and 12 were also conducted; the germination period was set up to 11 days.

Conclusions
In this work, the chemical investigation of the Rhodiola tibetica endophytic fungus Penicillium sp.HJT-A-6 led to the isolation and identification of a new quinazolinone alkaloid named peniquinazolinone A (1) and eleven known compounds.Compound 1 was identified through an extensive spectroscopic analysis and the modified Mosher's method.Compounds 2, 3, and 7 showed an optimal promoting effect on the seed germination of Rhodiola tibetica at a concentration of 0.01 mg/mL.Compounds 4 and 9 had optimal seed-germination-promoting activity at a concentration of 0.001 mg/mL, while the optimal concentration for compound 12 to promote Rhodiola tibetica seed germination was 0.1 mg/mL.Additionally, all the tested compounds assuredly delayed the seed germination of the host plant.Furthermore, the mechanism of these known compounds with seedgermination-promoting activity will be investigated using biochemical and transcriptomic methods.These above results not only broadened the structural diversity of quinazoline metabolites derived from fungi but also provided data support for understanding the interactive relationship between endophytic fungi and host plants.
and S9).The HMBC correlations from H-2 to C-6, from H-3 to C-1, from H-4 to C-6, from H-5 to C-3 and C-7, and from H-16 to C-7 and C-9 established the structural skeleton of quinazolinone.The proton spin systems of H-11/H-12/H-13/H-14/H-15 and H-13/13-OH, coupled with the HMBC correlations from H-11 to C-9, C-12, and C-13; from H-14 to C-13; and from H-15 to C-13 and C-14, confirmed the linkage of the quinazolinone group and the pentan-3-ol moiety at C-9.Consequently, the planar structure of compound 1 was constructed, named peniquinazolinone A.

Figure 6 .
Figure 6.Effects of all the tested compounds, 6-BA, and the blank control water on the seed germination period of Rhodiola tibetica.

Figure 6 .
Figure 6.Effects of all the tested compounds, 6-BA, and the blank control water on the seed germination period of Rhodiola tibetica.